Stroke and anoxic encephalopathy result in extensive morbidity and mortality. Although brain injury is triggered by ischemia, some ischemic neuronal damage develops during reperfusion and involves expression of new gene products. The design of interventions to limit brain injury during reperfusion is limited by the current understanding of molecular events in vivo. This project will determine the role of in vivo activation of mitogen activated protein kinase (MAPK) - related signaling pathways during reperfusion after cerebral ischemia. MAPK-related kinases mediate trophic factor and stress response induced gene expression by activating transcription factors, and may participate in the response to ischemia. The first series of studies will characterize the effect of cerebral ischemia and reperfusion on MAPK activation. These studies will employ a clinically relevant rat model of anoxic encephalopathy (asphyxial cardiac arrest) and of stroke (middle cerebral artery occlusion). MAPK-related kinase activation and substrate transcription factor activity will be quantified in brain homogenates. Localization of enzymes and intracellular distribution of enzymes will be examined using immunohistochemistry. Interventions known to mitigate neuronal injury (hypothermia) will be examined for their effects on MAPK. The second series of studies will examine the effects of drugs (geldanamycin and herbimycin A) that interfere with the proper functioning of the 90 kDa heat shock protein (hsp90), and that reduce levels of raf-1, a MAPK kinase kinase. The effects of these drugs on activation and intracellular localization of MAPK are unknown. For comparison, intracranial injections of MAPK kinase and MAPK inhibitors will be examined. The third series of studies will characterize the effect of hsp90/raf depletion and MAPK inhibitors on other proteins believed to be relevant to ischemic neuronal death as well as on neurological outcome after cerebral ischemia. Preliminary studies indicate that geldanamycin improves neurobehavioral recovery after asphyxial cardiac arrest. Thus, interventions in the MAPK cascade may have application in the development of neuroprotective agents. The conduct of this project will provide extensive opportunities for collaboration with other basic scientists at the University of Pittsburgh, and promote the candidate's growth as an independent researcher.